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Generation of induced neurons via direct conversion in vivo and in vitro

Torper, Olof LU (2014) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2014:20.
Abstract (Swedish)
Popular Abstract in Swedish

I min avhandling har jag tittat på möjligheten att göra om mänskliga hudceller

direkt till dopaminerga nervceller. Dessa uppvisar flera egenskaper tillskrivna de

dopaminerga nervceller vi hittar i hjärnan. Jag tittar även på möjligheten att transplantera

dessa som hudceller i hjärnan på råttor och sen göra om dom till nervceller

när de är på plats. Dessa celler bildar nervceller i samma utsträckning som när de

görs om i cellkultur. Sist riktar vi in oss på två olika celltyper i hjärnan som inte är

nervceller och gör om dessa direkt till nervceller på plats i hjärnan. Detta kan tillföra

ännu en dimension till cellterapi då nya celler... (More)
Popular Abstract in Swedish

I min avhandling har jag tittat på möjligheten att göra om mänskliga hudceller

direkt till dopaminerga nervceller. Dessa uppvisar flera egenskaper tillskrivna de

dopaminerga nervceller vi hittar i hjärnan. Jag tittar även på möjligheten att transplantera

dessa som hudceller i hjärnan på råttor och sen göra om dom till nervceller

när de är på plats. Dessa celler bildar nervceller i samma utsträckning som när de

görs om i cellkultur. Sist riktar vi in oss på två olika celltyper i hjärnan som inte är

nervceller och gör om dessa direkt till nervceller på plats i hjärnan. Detta kan tillföra

ännu en dimension till cellterapi då nya celler kan skapas på plats i hjärnan och

externa källor för celler inte behövs.

Våra kroppsceller innehåller identisk genetisk information i form av DNA packat

i 23 st kromosompar. Cellerna skiljer sig åt i vilken genetisk information som faktiskt

uttrycks genom olika kemiska modifikationer på kromosomerna. Dessa modifieringar

styrs av olika signaler, bla av särskilda proteiner kallade transkriptionsfaktorer.

Genom att tvinga en cell till att uttrycka vissa transkriptionsfaktorer kan

man ändra de kemiska modifikationerna och på så vis göra om cellen till en annan.

Det kallas cellulär omprogrammering. På så vis har man gjort om vanliga hudceller

till en ur-stamcell med förmågan att bilda alla de olika celler som utgör en individ.

Man har även lyckats att göra om en hudcell direkt till en nervcell.

Varför är då detta intressant? Förutom att det visar på våra cellers enorma kapacitet

att inta olika roller beroende på vilka gener som uttrycks så finns det ett

medicinskt intresse. I det som kallas cell-terapi ämnar man ersätta sjuka eller döda

celler med nya genom transplantation. Detta är speciellt intressant för hjärnan

eftersom dess förmåga att laga sig själv efter skador såsom stroke (Sv. slaganfall)

är begränsad. Många sjukdomar som drabbar det centrala nervsystemet är också

intressanta kandidater för cellterapi. I Parkinsons sjukdom (PD) dör en specifik

sorts nervceller kallade dopaminerga nervceller vilka frisätter signalsubstansen

dopamin i hjärnan. Detta leder till svårigheter att utföra medvetna handlingar och

muskulär stelhet. Det finns idag ingen beständig behandling för patienter med PD

och de mediciner som ges tappar i effekt med tiden och besvärliga biverkningar

uppstår. Försök med patienter med PD till vilka man transplanterat fetal vävnad

innehållande de celler som så småningom bildar de dopminerga nervcellerna har

visat att det går att lindra symptomen förknippade med PD. Tillgången på fetal

vävnad är dock begränsad och det råder en del etiska kontroverser kring hanteringen

av den. En annan typ av celler man undersöker är embryonala stam celler.

Dessa kan isoleras från ett tidigt stadium av det befruktade ägget och har förmågan

att bilda alla celler i kroppen. Genom att styra dom till att bli dopaminerga nervceller

skulle de kunna användas i transplantationer till patienter med PD. Det finns

dock risker assoccierade med tumörbildningar i användandet av dessa celler då de

aktivt delar sig samt att vissa etiska kontroverser återstår.

20

Hudceller som direkt görs om till nervceller kringgår flera av dessa problem.

De är enkla att isolera från individen och eftersom de inte passerar ett stamcells

stadie utgör de ingen risk i bildandet av tumörer. Eftersom cellerna kan tas direkt

från patienten själv begränsar man även risken för bortstötning då cellerna är immunomatchade

till individen. (Less)
Abstract
Cellular reprogramming is when one cell is changed into another. This involves structural modifications on the DNA of a cell

resulting in a transcriptional change. This occurs naturally during development when early pluripotent cells gradually differentiate into more

specialized cells that finally result in a complete organism. This is a finely orchestrated event that includes both extrinsic and intrinsic signaling.

Cellular reprogramming can be induced artificially by exposing a somatic cell to a foreign microenvironment or by the forced expression

of various transcription factors. Recent studies have shown the possibility to revert a somatic cell back into a pluripotent stem cell, termed

... (More)
Cellular reprogramming is when one cell is changed into another. This involves structural modifications on the DNA of a cell

resulting in a transcriptional change. This occurs naturally during development when early pluripotent cells gradually differentiate into more

specialized cells that finally result in a complete organism. This is a finely orchestrated event that includes both extrinsic and intrinsic signaling.

Cellular reprogramming can be induced artificially by exposing a somatic cell to a foreign microenvironment or by the forced expression

of various transcription factors. Recent studies have shown the possibility to revert a somatic cell back into a pluripotent stem cell, termed

induced pluripotent stem cell (iPS) or directly to a different somatic cell using this strategy.

In this thesis I focus on the direct reprogramming where one terminally differentiated cell is directly converted into another without passing

a pluripotent state. Using lentiviral vectors we could convert embryonic and postnatal human fibroblasts into functional neurons (iN) by the

forced expression of Ascl1, Brn2 and Myt1L (ABM). By including the additional factors, Foxa2 and Lmx1a, subtype specific neurons could be

obtained that release dopamine, express specific markers and exhibit electrophysiological properties characteristic of dopaminergic neurons.

Further we show the possibility to transplant fibroblasts and astrocytes into brains of adult rats and then convert them into neurons in vivo.

These cells expressed pan- neuronal markers and converted at similar rates as reported in vitro. Using Cre inducible lentiviral vectors, coding

for ABM and inject these into the brains of transgenic mice expressing Cre under the GFAP promoter, we could specifically target astrocytes and

convert these into neurons in vivo. Using the same strategy we cloned the three factors, Ascl1, Lmx1a and Nurr1 (ALN) together with GFP, into

Cre inducible recombinant adeno associated viral vectors (rAAV) with the aim to convert NG2 glia into dopaminergic neurons. rAAV vectors

are interesting tools for clinical applications because of their low pathogenicity and their ability to infect both dividing and non-dividing cells.

By including a synapsin promoter for the GFP reporter we could specifically visualize converted cells that expressed the pan neuronal markers

NeuN and MAP2 but failed to induce a dopaminergic phenotype. More studies aim to study these cells after a longer maturation time and their

functional properties in terms of electrophysiology and synaptic formation.

Cellular reprogramming of somatic cells is an interesting option to previously studied sources in cell replacement therapies that often are

associated with logistical and ethical concerns. They are readily available cells that can be obtained from the skin of a patient and direct conversion

offers further advantages over iPS cells as they are non-proliferating cells eliminating the risk of forming tumors when transplanted.

Further, in vivo reprogramming offers an alternative to traditional cell therapy by creating new neurons in the brain removing the need of an

exogenous cell source. The brain is of particular interest for cell replacement therapies as its capacity to repair itself after injuries like stroke

is limited and treatments for neurological disorders like Parkinson’s disease (PD) progressively decline in effectiveness and are associated

with severe side effects.

In summary, this thesis shows the possibility to directly convert human, adult fibroblasts into functional dopaminergic neurons by the forced

expression of transcription factors important in neural development. We further show the possibility to transplant fibroblasts and astrocytes

into the brains of rats and convert them into neurons in situ. We also show the possibility to convert two types of glia cells, astrocytes and NG2

glia residing in the brain into neurons by using transgenic mice and Cre inducible vectors. This could also be done by using a rAAV vector commonly

used in clinical trials. Future studies should focus on factors involved in the specificity of the required cell and how well the cell that is

formed correspond genetically, functionally and viably to its endogenous counterpart. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Hermansson, Ola, Department of Neuroscience, Karolinska institutet, Retzius väg 8 S-171 77 Stockholm, Sweden
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Cellular reprogramming, cell replacement therapies, induced dopaminergic neurons, in vivo reprogramming, Lentiviral vectors, AAV vectors
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2014:20
pages
125 pages
publisher
Neurobiology
defense location
Segerfalksalen, Wallenberg Neuroscience Center, Lund, Sweden
defense date
2014-02-28 13:00
ISSN
1652-8220
ISBN
978-91-87651-44-1
language
English
LU publication?
yes
id
b3b72c69-3e85-4faa-b9d7-803e085aa7c0 (old id 4276198)
date added to LUP
2014-01-29 17:04:00
date last changed
2016-09-19 08:44:48
@phdthesis{b3b72c69-3e85-4faa-b9d7-803e085aa7c0,
  abstract     = {Cellular reprogramming is when one cell is changed into another. This involves structural modifications on the DNA of a cell<br/><br>
resulting in a transcriptional change. This occurs naturally during development when early pluripotent cells gradually differentiate into more<br/><br>
specialized cells that finally result in a complete organism. This is a finely orchestrated event that includes both extrinsic and intrinsic signaling.<br/><br>
Cellular reprogramming can be induced artificially by exposing a somatic cell to a foreign microenvironment or by the forced expression<br/><br>
of various transcription factors. Recent studies have shown the possibility to revert a somatic cell back into a pluripotent stem cell, termed<br/><br>
induced pluripotent stem cell (iPS) or directly to a different somatic cell using this strategy.<br/><br>
In this thesis I focus on the direct reprogramming where one terminally differentiated cell is directly converted into another without passing<br/><br>
a pluripotent state. Using lentiviral vectors we could convert embryonic and postnatal human fibroblasts into functional neurons (iN) by the<br/><br>
forced expression of Ascl1, Brn2 and Myt1L (ABM). By including the additional factors, Foxa2 and Lmx1a, subtype specific neurons could be<br/><br>
obtained that release dopamine, express specific markers and exhibit electrophysiological properties characteristic of dopaminergic neurons.<br/><br>
Further we show the possibility to transplant fibroblasts and astrocytes into brains of adult rats and then convert them into neurons in vivo.<br/><br>
These cells expressed pan- neuronal markers and converted at similar rates as reported in vitro. Using Cre inducible lentiviral vectors, coding<br/><br>
for ABM and inject these into the brains of transgenic mice expressing Cre under the GFAP promoter, we could specifically target astrocytes and<br/><br>
convert these into neurons in vivo. Using the same strategy we cloned the three factors, Ascl1, Lmx1a and Nurr1 (ALN) together with GFP, into<br/><br>
Cre inducible recombinant adeno associated viral vectors (rAAV) with the aim to convert NG2 glia into dopaminergic neurons. rAAV vectors<br/><br>
are interesting tools for clinical applications because of their low pathogenicity and their ability to infect both dividing and non-dividing cells.<br/><br>
By including a synapsin promoter for the GFP reporter we could specifically visualize converted cells that expressed the pan neuronal markers<br/><br>
NeuN and MAP2 but failed to induce a dopaminergic phenotype. More studies aim to study these cells after a longer maturation time and their<br/><br>
functional properties in terms of electrophysiology and synaptic formation.<br/><br>
Cellular reprogramming of somatic cells is an interesting option to previously studied sources in cell replacement therapies that often are<br/><br>
associated with logistical and ethical concerns. They are readily available cells that can be obtained from the skin of a patient and direct conversion<br/><br>
offers further advantages over iPS cells as they are non-proliferating cells eliminating the risk of forming tumors when transplanted.<br/><br>
Further, in vivo reprogramming offers an alternative to traditional cell therapy by creating new neurons in the brain removing the need of an<br/><br>
exogenous cell source. The brain is of particular interest for cell replacement therapies as its capacity to repair itself after injuries like stroke<br/><br>
is limited and treatments for neurological disorders like Parkinson’s disease (PD) progressively decline in effectiveness and are associated<br/><br>
with severe side effects.<br/><br>
In summary, this thesis shows the possibility to directly convert human, adult fibroblasts into functional dopaminergic neurons by the forced<br/><br>
expression of transcription factors important in neural development. We further show the possibility to transplant fibroblasts and astrocytes<br/><br>
into the brains of rats and convert them into neurons in situ. We also show the possibility to convert two types of glia cells, astrocytes and NG2<br/><br>
glia residing in the brain into neurons by using transgenic mice and Cre inducible vectors. This could also be done by using a rAAV vector commonly<br/><br>
used in clinical trials. Future studies should focus on factors involved in the specificity of the required cell and how well the cell that is<br/><br>
formed correspond genetically, functionally and viably to its endogenous counterpart.},
  author       = {Torper, Olof},
  isbn         = {978-91-87651-44-1},
  issn         = {1652-8220},
  keyword      = {Cellular reprogramming,cell replacement therapies,induced dopaminergic neurons,in vivo reprogramming,Lentiviral vectors,AAV vectors},
  language     = {eng},
  pages        = {125},
  publisher    = {Neurobiology},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {Generation of induced neurons via direct conversion in vivo and in vitro},
  volume       = {2014:20},
  year         = {2014},
}